Protocols

Everything

about labeling

Immunolabeling

abberior offers protocols for immunolabeling of cultured mammalian cells (adherent) via two different procedures – immunolabeling cytoskeletal elements (methanol fixation) and immunolabeling membrane bound organelles (formaldehyde fixation).

You can download the abberior immunolabeling recommendations here:

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Labeling of mammalian living cells

abberior offers a labeling protocol for labeling of lysosomes (pepstatin), actin (jasplakinolide) or tubulin (cabazitaxel) in adherent mammalian living cells using abberior LIVE probes.

You can download the abberior live-cell labeling protocol here:

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Antibody labeling

abberior offers labeling protocols for labeling of antibodies via two different procedures:

  • labeling of amine-reactive abberior labels

  • labeling of thiol-reactive abberior labels.

Degree of labeling (DOL)

The degree of labeling (DOL) is the average number of dye molecules coupled to a protein molecule (e.g. an antibody). The DOL can be determined from the absorption spectrum of the labeled antibody.

Procedure

For the DOL determination, the antibody conjugate solution should be gel filtrated. Then, the UV/VIS spectrum of the solution is acquired in a window starting at ~250 nm and covering the complete absorbance band of the coupled dye (see the dye data sheet provided by Abberior). The absorption spectra must be acquired using a UV transparent quartz cuvette. Read the following values from the absorption spectrum:

λmax: the wavelength where the absorbance has its maximum (should be close to the absorbance maximum of the pure dye, compare the respective data sheet)
Amax: the absorbance at λmax
A280: the absorbance at 280 nm

Analysis

The DOL is calculated from the measured values according to the following equation:

Here, the symbols denote:

Aprot: absorbance of the pure protein at 280 nm (absorption maximum of proteins)
εprot: molar extinction coefficient (in M-1cm-1) of the pure protein at 280 nm
εmax:  molar extinction coefficient (in M-1cm-1) of the dye at its absorbance maximum. For Abberior dyes, εmax is specified in the respective product data sheet.
C280: correction factor which depends on the dye’s spectral properties. For Abberior dyes, C280 is specified in the dye’s data sheet.
εprot: molar extinction coefficient (in M-1cm-1) of the pure protein at 280 nm. Unless specified by the vendor of the antibody it must be determined in a separate measurement.

Note that the above equation is only accurate if the extinction coefficient of the free dye εmax at the maximum absorbance is equal to the value of the dye-antibody conjugate at its maximum absorbance. In practice, the calculated DOL values may be off by ~20 %.